The overall goal of this project is to continue to characterize an endogenous inhibitor of intracellular proteolysis in reticulocytes which appears important in degrading abnormal hemoglobins and red blood cell survival. We have recently purified the inhibitor to homogeniety and shown that it selectively blocks the activity of non-lysosomal high molecular weight and calcium-dependent proteases. Based upon experiments with crude fractions, we believe that the inhibitor is repressed in cells by an ATP- dependent mechanism involving ubiquitin. With aging of erythroid cells, there is a marked loss of ATP-dependent proteolytic activity which appears to be a result of an inability to repress the inhibitor rather than a loss of protease per se. We will attempt to demonstrate that ubiquitin can directly or indirectly regulate protease/inhibitor interaction and that the isolated high molecular weight and calcium-dependent proteases are part of a larger ATP- dependent complex. Such experiments will follow complex formation of 125I-labelled inhibitor with proteases using gel chromatography. We are also attempting to generate suitable monoclonal antibodies to the proteases and inhibitor which will be utilized to probe interacting of these components. In addition the role of the ATP-dependent proteases and inhibitor in the full ATP-dependent system will be studied by removal or inactivation of components by antibody binding. Further characterization of the inhibitor will be accomplished by isolating active fragments using FPLC. Such studies should lead to identification of domains involved in inhibition of each protease. Finally, monoclonal antibodies will also be used to map functional inhibitor domains and to define fragments. These studies should lead to further understanding of the energy requirement for proteolysis in cells. In addition, understanding this proteolytic system could give insight into modulation of excess globin chains in thalassemias and accumulation of abnormal globins in certain hemoglobinopathies.